PoROvVTIDi[unreadable]ED. In eukaryotes, the 'multivesicular body' (MVB) pathway delivers transmembrane proteins and lipids into the lumen of the lysosome for degradation. As a consequence, MVBs are essential for regulating cell surface protein composition and maintaining lysosomal function. Numerous cellular functions, such as nutrient uptake, cell communication and immune response are dependent on MVBs. The MVB sorting machinery performs a unique membrane budding event, which results in the formation of vesicles into the lumen of the endosome. Retroviruses such as HIV co-opt the MVB machinery during viral infection to complete formation of viral particles via a similar membrane budding event at the plasma membrane. Therefore, the MVB sorting machinery has been recognized as a target for the development of new drugs combating HIV infection, a disease that kills more than 2 million people per year worldwide. Several multiprotein complexes, called the ESCRTs, execute cargo sorting and vesicle formation at the MYB. To perform their function, the soluble ESCRT complexes are recruited from the cytoplasm and sequentially assemble on the endosomal membrane where they sort ubiquitinated cargo into forming vesicles. To complete vesicle formation, the ESCRT complexes are disassembled by the activity of the AAA-type ATPase Vps4. Without Vps4 function, the ESCRT machinery remains on the membrane and MVB vesicle formation is inhibited. Up until now, it had been thought that the disassembly function of Vps4 was a constitutive process. However, our preliminary studies have identified three proteins that regulate the activity of Vps4 on several levels. Vtal appears to enhance the disassembly reaction that stimulates Vps4 ATPase,activity. Ftil works together with Vtal in the activation of Vps4. Istl interferes with Vps4 function and Istl activity itself appears to be regulated by its stability. Based on these findings we propose a revised model in which the Vps4-dependent disassembly of the ESCRT complexes represents a key regulatory step within the MVB pathway.We propose thateukaryotic cells modulate the activity of the MVB pathway by regulating the degradation rate of Istl, thereby regulating the activity of Vps4.